E-cadherin accumulation within the lymphovascular embolus of inflammatory breast cancer is due to altered trafficking.

E-Cadherin functions as a tumor suppressor in some invasive breast carcinomas and metastasis is promoted when its expression is lost. It has been observed, however, that in one of the most aggressive human breast cancers, inflammatory breast cancer (IBC), E-cadherin is overexpressed and this accounts for the formation of the lymphovascular embolus, a structure efficient at metastasis and resistant to chemotherapy through unknown cytoprotective mechanisms. Studies using a human xenograft model of IBC, MARY-X, indicate that the mechanism of E-cadherin overexpression is not transcriptional but related to altered protein trafficking. By real-time RT-PCR, E-cadherin transcript levels in MARY-X were 3- to 11-fold less than in other E-cadherin positive human breast carcinoma lines but the protein levels were 5- to 10-fold greater. In addition, several smaller E-cadherin protein fragments, e.g. 95 kDa, were present. To explain these observations, it was hypothesized that there may be altered protein trafficking. A real-time RT-PCR screen of candidate molecules generally known to regulate protein trafficking was conducted. The screen revealed 3.5- to 7-fold increased ExoC5 level and 10 to 20 fold decreased HRS and RAB7 levels, which was confirmed in human microdissected lymphovascular emboli. Since these alterations may only be correlative with E-cadherin overexpression, one of the molecules, Rab7, was selectively knocked down in MCF-7 cells. An increase in the full length 120 kDa E-cadherin and the de novo appearance of the 95 KD band were observed. These findings suggest that it is the altered E-cadherin trafficking that contributes to its oncogenic rather than suppressive role in IBC.

Breast carcinomatous tumoral emboli can result from encircling lymphovasculogenesis rather than lymphovascular invasion.

The canonical view of the origin of tumor lymphovascular emboli is that they usually originate from lymphovascular invasion as part of a multistep metastatic process. Recent experimental evidence has suggested that metastasis can occur earlier than previously thought and we found evidence that tumor emboli formation can result from the short-circuiting step of encircling lymphovasculogenesis. Experimentally, we used a xenograft of human inflammatory breast cancer (MARY-X), a model that exhibited florid tumor emboli, to generate tumoral spheroids in vitro. In observational studies, we chose human breast carcinoma cases where there appeared to be a possible transition of in situ carcinoma to lymphovascular emboli without intervening stromal invasion. These cases were studied by morphometry as well as IHC with tumor proliferation (Ki-67) and adhesion (E-cadherin) markers, myoepithelial (p63), as well as endothelial (podoplanin [D2-40], CD31, VEGFR-3, Prox-1) markers. Unlabelled spheroids coinjected with either GFP or RFP-human myoepithelial cells or murine embryonal fibroblasts (MEFs) gave rise to tumors which exhibited GFP/RFP immunoreactivity within the cells lining the emboli-containing lymphovascular channels. In vitro studies demonstrated that the tumoral spheroids induced endothelial differentiation of cocultured myoepithelial cells and MEFs, measured by real time PCR and immunofluorescence. In humans, the in situ clusters exhibited similar proliferation, E-cadherin immunoreactivity and size as the tumor emboli (p =.5), suggesting the possibility that the latter originated from the former. The in situclusters exhibited a loss (50%-100%) of p63 myoepithelial immunoreactivity but not E-cadherin epithelial immunoreactivity. The tumor emboli were mainly present within lymphatic channels whose dual p63/CD31, p63/D2-40 and p63/VEGFR-3 and overall weak patterns of D2-40/CD31/VEGFR-3 immunoreactivities suggested that they represented immature and newly created vasculature derived from originally myoepithelial-lined ducts. Collectively both experimental as well as observational studies suggested the possibility that these breast cancer emboli resulted from encircling lymphovasculogenesis rather than conventional lymphovascular invasion.